Recently, I did sample digestion of my protein standard using FASP and I found that it had significantly more methionine oxidation than previous samples. Now, I am looking for any leads as to minimize this. Any comments or suggestions are welcome. Thank you.
Hi,
It is important to minimize exposure of samples to air and elevated temperatures to limit artefactual methionine oxidation, so work fast and consistently :).
In case you continue to encounter increased levels of methionine oxidation, you can also add L-methionine to your samples.
Hi Aaron James Reyes,
We recently published a paper for the handling of peptides, and we did a chapter on oxidation that might be of interest for you! I hope this will help.
Article Tutorial review for peptide assays: An ounce of pre-analytic...
You may use antioxidants to inhibit the oxidation. For this, catalase, and methionine are typically used but you may add water-soluble antioxidants such as phenolic acids can also do the job. FASP is inherently an extended protocol and smaller sample volumes, and larger cut-off values of the membrane can reduce the required uptime. Thus, the whole process can be concluded relatively faster to limit met oxidation.
BTW, you may also look for S-TRAP as an alternative way of SDS-removal
and on-membrane cleavage technique. I did not compare but papers declare it has the same efficiency as FASP but completes the process faster...
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